Autophagy derivative

Abstract

To provide a skin-whitening agent, autophagy derivative, and epidermis hyperplasia inhibitor.SOLUTION: Provided are a humus extract containing fulvic acid and mineral, and / or a skin-whitening agent including uncoupler other than fulvic acid, an autophagy derivative including uncoupler in keratinocyte, and an epidermis hyperplasia inhibitor including uncoupler.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to , O -autophagy induction agent .

Background Art

[0002] Autophagy is a general term for a system that degrades a part of the cytoplasm, such as cytoplasmic components containing proteins and intracellular organelles such as mitochondria, and plays a role such as decomposing proteins under starvation conditions to supply amino acids. Autophagy is a series of processes in which an isolation membrane generated from the rough endoplasmic reticulum-mitochondria contact site in the cell takes in a substrate, forms a double-membrane structure called an autophagosome, fuses with a lysosome, becomes an autophagolysosome with a single-membrane structure, and digestive enzymes are supplied to decompose the internal substrate. Autophagy is classified into macroautophagy, microautophagy, and chaperone-mediated autophagy, and autophagy usually refers to macroautophagy. It has been reported that autophagy is involved in the determination of skin color by regulating melanosome digestion in keratinocytes (Non-Patent Document 1).

[0003] Freckles, age spots, and skin pigmentation after sunburn are caused by enhanced melanin production due to the activation of melanocytes present in the skin, and are one of the skin problems in middle-aged and elderly people. Generally, melanin is considered to be formed by the action of tyrosinase, an enzyme biosynthesized in pigment cells, and various whitening components that exhibit the effect of suppressing melanin production by inhibiting tyrosinase activity have been proposed.

[0004] Humus is mainly a polymer formed by the condensation of the decomposition products of carbohydrates and proteins in plant residues, microbial remains, etc. in the soil by microorganisms over a long period of time. Humus contains amorphous polymers such as fulvic acid and humic acid. Humic acid is a reddish-brown or black-brown amorphous high-molecular organic acid that is soluble in alkali and insoluble in acid. On the other hand, fulvic acid is an amorphous high-molecular organic acid that is soluble in both alkali and acid. It is known that in addition to fulvic acid and humic acid, humus also contains fatty acids, organic acids, amino acids, proteins, minerals, etc.

[0005] Thus, fulvic acid (FA: Fulvic acid) is an organic acid present in humus. In some parts of the United States and in Saga Prefecture, Japan, for example, humus containing fulvic acid is recognized, and the standardization of fulvic acid in industry is being promoted.

[0006] Also, in Patent Document 1, the present inventors reported that fulvic acid has a remarkable effect on lifestyle-related diseases, improves liver function, and fulvic acid acts as a decoupling agent.

[0007] Regarding humic acid, for example, the anti-inflammatory effect has been reported in Non-Patent Document 2.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present invention , O -autophagy induction agent is provided.

Means for Solving the Problems

[0011] As a result of intensive studies to achieve the above object, the present inventors have obtained the finding that fulvic acid has an effect of activating autophagy in keratinocytes. Furthermore, based on the activation of autophagy by such fulvic acid, it has been found that the digestive activity of melanosomes is activated in keratinocytes, and in particular, fulvic acid containing minerals has excellent activity. It has also been found that the digestive activity of melanosomes in keratinocytes can be further activated by combining fulvic acid with a decoupling agent other than fulvic acid.

[0012] The present invention has been completed through further studies based on these findings, and further O of -autophagy induction agent is provided.

[0013] An autophagy inducer comprising a composition containing fulvic acid according to item 1. The autophagy inducer according to item 1, which induces autophagy in keratinocytes. The autophagy inducer according to item 1 or 2, which is used for whitening and / or suppressing epidermal hyperplasia. The autophagy inducer according to any one of items 1 to 3, wherein the composition containing fulvic acid has a decoupling effect.

Effects of the Invention

[0014] Fulvic acid has a significantly excellent autophagy activation effect, as well as a whitening effect and an epidermal hyperplasia inhibitory effect based on the autophagy activation effect, so , O -autophagy induction of the agent it is useful as an active ingredient.

Brief Description of the Drawings

[0015] [Figure 1] It is a graph showing the effect of an autophagy inhibitor on the melanosome digestion activity taken up by keratinocytes. n = 8. The data shows the mean ± standard error value after subtracting the absorbance in the wells without added melanosomes (Blank). One-way ANOVA. ***: P < 0.005 vs CRL, : P < 0.005 vs Hu. [Figure 2] It is a graph showing the additive effect of metformin (MET), which is an uncoupling agent, and humic on the melanosome digestion activity taken up by keratinocytes. n = 6. The data shows the mean ± standard error value after subtracting the absorbance in the wells without added melanosomes (Blank). One-way ANOVA. ***: P < 0.005 vs CRL. [Figure 3] It is a graph showing the synergistic effect of fulvic acid and minerals on the melanosome digestion activity taken up by keratinocytes. n = 6. The data shows the mean ± standard error value after subtracting the absorbance in the wells without added melanosomes (Blank). One-way ANOVA. ***: P < 0.005 vs CRL.

Mode for Carrying Out the Invention

[0016] Hereinafter, embodiments of the present invention will be described in detail.

[0017] In this specification, "containing, comprise" includes the meaning of "essentially consisting of" and the meaning of "consisting of only".

[0018] This Induction of autophagy of the invention agent is , a composition containing fulvic acid characterized by including.

[0019] An uncoupling agent refers to a compound that inhibits the coupling of the electron transport system and the ATP synthesis reaction in the oxidative phosphorylation of organisms. In the present invention, known uncoupling agents can be used without particular limitation as the uncoupling agent. Such uncoupling agents include, for example, those containing fulvic acid composition , metformin, buformin, phenformin, and the like. In the present invention, the uncoupling agent can be used alone or in combination of two or more.

[0020] Fulvic acid is an amorphous polymer organic acid that is soluble in both alkali and acid among the substances contained in humus. The humus used as the raw material for fulvic acid is not particularly limited in terms of production area, state, etc., and any humus can be used.

[0021] Examples of such humus include humic shale obtained from an ancient plant deposit (shale layer) in Emery County, Utah, USA. Humic shale is what remains as an organic substance without being transformed into oil and coal from the fertile soil containing many nutrients about 700 million to 127 million years ago. The plants that lived in the said era were buried underground when entering the ice age, changed into peat moss by microorganisms such as bacteria, new organic substances were piled up on it, and were decomposed again by bacteria and the like, forming a sedimentary layer over time. The humus contained in the formed sedimentary layer mainly consists of substances composed of organic polymer polymers such as fulvic acid and humic acid, and contains more than 70 kinds of trace minerals.

[0022] The method for extracting fulvic acid from humus is not particularly limited, and various known methods can be used. Examples of the extraction means include solid-liquid extraction, liquid-liquid extraction, immersion, leaching, decoction, reflux extraction, supercritical fluid extraction, microwave extraction, mixing and stirring, etc. The extraction means can be carried out alone or in combination of two or more.

[0023] When extracting fulvic acid by mixing humus with an extraction solvent, examples of the extraction solvent include water (e.g., distilled water, ion-exchanged water, reverse osmosis water, etc.), alcohol (e.g., methanol, ethanol, propanol, etc.), and mixtures thereof. The extraction time, extraction temperature, pH of the extraction solvent, etc. in the extraction operation can be appropriately set in consideration of the extraction efficiency, etc.

[0024] For the extract of humus, filtration may be further performed for removing impurities and sterilization. The means of filtration is not particularly limited and can be carried out by conventional methods.

[0025] For the extract of humus, a treatment for isolating or purifying fulvic acid can be further performed. Examples of such isolation or purification treatments include concentration, vacuum concentration, distillation, fractional distillation, phase transfer, solvent extraction, crystallization, recrystallization, chromatography, etc. These methods can be carried out alone or in combination of two or more.

[0026] What is used in the present invention a composition containing fulvic acid as , F is not particularly limited as long as it contains fulvic acid, preferably fulvic acid and minerals. Also, what is used in the present invention a composition containing fulvic acid can be produced by the above method.

[0027] a composition containing fulvic acid The pH of a composition containing fulvic acid is in the acidic range, for example, pH 1 to 6, preferably pH 2 to 5, particularly pH 3.

[0028] a composition containing fulvic acid As

[0029] It is known that in addition to fulvic acid and humic acid, the extract of humus contains fatty acids, organic acids, amino acids, proteins, minerals, etc. a composition containing fulvic acid When using a crude extract of humus as a composition containing fulvic acid in the present invention, in addition to fulvic acid, it may contain fatty acids, organic acids, amino acids, proteins, minerals, etc., particularly minerals. Since fulvic acid can chelate minerals, a composition containing fulvic acid when minerals are contained in a composition containing fulvic acid , the fulvic acid may be in a state of chelating the minerals. Thus,

[0030] by containing minerals in addition to fulvic acid, the whitening effect is improved.

[0031] a composition containing fulvic acid The form of

[0032] a composition containing fulvic acid is not particularly limited, and examples include powdery, granular, solid, solution, suspension, emulsion, concentrated solution, slurry, etc.

[0032] a composition containing fulvic acid Either a self-prepared product or a commercially available product can be used. a composition containing fulvic acid Examples of commercially available products of -1It has an infrared absorption peak (see Japanese Patent No. 6120342). Further, the average molecular weight of the fulvic acid contained in the humic substance is usually less than 4,000, preferably 3,500 or less, particularly preferably 3,000 or less, and more preferably 1,000 to 3,000. The humic substance contains 90% by mass or more of minerals, and the content of fulvic acid is less than 10% by mass.

[0033] The autophagy inducer of the present invention may further contain a deconjugating agent other than fulvic acid, and by combining fulvic acid and a deconjugating agent other than fulvic acid in this way, the whitening effect can be further improved.

[0034] The present invention O of - autophagy induction agent contained a composition containing fulvic acid Examples of the ratio include 0.0001 to 99% by mass, 0.001 to 80% by mass, 0.01 to 70% by mass, and the like.

[0035] The present invention O of - autophagy induction agent is , for skin care, etc. It can be used as cosmetics, food and drink (particularly food and drink for the purpose of health care, health maintenance, promotion, etc. (for example, health food, functional food, nutritional composition, dietary supplement, supplement, health food, food for specified health use, food with nutritional function, or food with functional indication)), quasi-drugs, pharmaceuticals, etc. Further, the present invention O of - autophagy induction agent is , whitening effect, autophagy induction and activation It also includes the meaning of additives that impart an effect and an epidermis thickening inhibitory effect.

[0036] The above-mentioned food and drink include a composition containing fulvic acid It can also be used as it is, and if necessary, excipients, vitamins, minerals, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, coloring agents, flavors, stabilizers, preservatives, sustained-release regulators, surfactants, brighteners, solvents, wetting agents, etc. can be formulated.

[0037] Food and drink products include all food and drink products that can be ingested by mammals (including humans), such as dairy products; fermented foods (such as yogurt, cheese, etc.); beverages (such as coffee, juice, cocoa, tea beverages, sports drinks, nutritious drinks like soft drinks, milk drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, Japanese sake, Western liquor, fruit wine, etc.); spreads (such as custard cream, etc.); pastes (such as fruit paste, etc.); Western confectionery (such as chocolate, donuts, pies, cream puffs, gums, gummies, jelly, candies, cookies, cakes, puddings, biscuits, etc.); frozen confectionery (such as ice cream, ice candy, sherbet, etc.); foods (such as curry, beef bowl, miso soup, soup, meat sauce, pasta, pickles, jam, ham, sausage, bacon, etc.); seasonings (such as dressing, furikake, umami seasonings, soup stock, miso, soy sauce, sauce, ketchup, oyster sauce, etc.).

[0038] The manufacturing method of food and drink products is not particularly limited, and it can be carried out according to known methods as appropriate.

[0039] The dosage unit form when used as a supplement is not particularly limited and can be appropriately selected. For example, tablets, capsules, granules, liquids, powders, etc. can be mentioned.

[0040] The intake amount of food and drink products can be appropriately set according to various conditions such as the weight, age, gender, and symptoms of the consumer.

[0041] For the above-mentioned pharmaceuticals, a composition containing fulvic acid only can be used, or it can also be used by mixing with other pharmaceutical ingredients described in the Japanese Pharmacopoeia such as vitamins and crude drugs.

[0042] The present invention O of - autophagy induction agent When prepared as a pharmaceutical, a composition containing fulvic acid it can be prepared together with a non-toxic carrier, diluent or excipient acceptable in pharmaceuticals into tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (powders), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, injections (including cases where it is formulated as a liquid by mixing with distilled water or infusion solutions such as amino acid infusion or electrolyte infusion during use), etc., to form pharmaceutical preparations.

[0043] The method of administering the pharmaceutical is not particularly limited and can be carried out, for example, by transdermal administration, oral administration, intra-arterial administration, intravenous administration, intraoral administration, rectal administration, enteral administration, etc.

[0044] The dosage of the pharmaceutical can be appropriately determined according to various conditions such as the patient's weight, age, gender, symptoms, etc.

[0045] In addition to the above cosmetics, a composition containing fulvic acid components usually used in cosmetics, such as whitening agents, moisturizing agents, antioxidants, oily components, ultraviolet absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, etc., can be appropriately formulated as needed.

[0046] Cosmetics include all cosmetics applicable to the skin, mucous membranes, body hair, hair, scalp, nails, teeth, facial skin, lips, etc. of animals (including humans).

[0047] The dosage forms of cosmetics can adopt a wide range of dosage forms, such as aqueous solution systems, solubilized systems, emulsion systems, powder systems, oil liquid systems, gel systems, ointment systems, aerosol systems, water-oil two-layer systems, water-oil-powder three-layer systems, etc.

[0048] The use of the cosmetics is also arbitrary. For example, in the case of basic cosmetics, there are facial cleansers, lotions, milks, creams, gels, essences, beauty liquids, packs, masks, etc. In the case of makeup cosmetics, there are foundations, lipsticks, blushes, eyeshadows, eyeliners, mascaras, etc. In the case of nail cosmetics, there are manicures, base coats, top coats, cuticle removers, etc. In addition, there are facial cleansers, (paste or liquid) dentifrices, mouthwashes, massage agents, cleansing agents, aftershave lotions, pre-shave lotions, shaving creams, body soaps, soaps, shampoos, rinses, hair treatments, hair styling agents, hair tonics, hair growth agents, antiperspirants, bath salts, etc.

[0049] The present invention O of -autophagy induction agent is is applied to mammals including humans (preferably humans).

[0050] In addition, quasi-drugs in the pharmaceuticals and cosmetics in the present invention are also included.

[0051] As shown in the test examples described below, the inventors , F found that furoic acid activates autophagy in keratinocytes. Furthermore, based on the activation effect of the autophagy, it was observed that furoic acid activates the digestive activity of melanosomes in keratinocytes, so that a whitening effect can be expected. Thus, furoic acid has a whitening effect such as lightening of post-sun pigmentation, freckles, chloasma, erythema, etc., making dullness less noticeable, and can maintain healthy skin. Therefore, it is useful as a whitening cosmetic, a skin care cosmetic, etc.

[0052] It is known that a phenotype characterized by epidermal thickening is shown due to the deficiency of autophagy-related genes (Biochem Biophys Res Commun. 2013 Jan 11;430(2):689-94., J Dermatol Sci. 2013 Jul;71(1):67-75., Exp Dermatol. 2018 Oct;27(10):1142-1151.). Thus, since it is known that the epidermis thickens when autophagy does not occur, fullvoic acid has an autophagy activation effect in keratinocytes, and thus an inhibitory effect on epidermal thickening can be expected.

[0053] Therefore , F Ruvo acid is has a remarkably excellent autophagy activation effect, as well as a whitening effect and an epidermal thickening inhibitory effect based on the autophagy activation effect , O -autophagy induction of the agent can be suitably used as an active ingredient. Containing fullvoic acid and minerals composition has an even better whitening effect . In addition, by combining fullvoic acid with a decoupling agent other than fullvoic acid, the whitening effect can be further improved.

[0054] In addition, since fullvoic acid is a natural-derived component, it has high safety.

Examples

[0055] Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples and the like.

[0056] Test Example 1 pMRX-IP-GFP-LC3-RFP (Addgene, #84573) was subcloned into the pMXs-puro retroviral vector. Using this, HaCaT cells stably expressing GFP-LC3-RFP were established by drug selection with 4 μg / ml puromycin, and autophagy was measured using these cells. In the experiment, MEM essential amino acid solution (50×) (WAKO) was added to autophagy dedicated medium (WAKO, Osaka, Japan) containing 20% fetal bovine serum, 10 ng / ml hrEGF and antibiotics to a final concentration of 1× (1×AA) (hereinafter referred to as autophagy induction medium), and a humidifier was added simultaneously. After culturing the cells in this medium, the cells were collected, and the fluorescence intensities of GFP and RFP were measured with a flow cytometer.

[0057] From the results of this experiment, it was confirmed that autophagy was enhanced in a concentration-dependent manner by the addition of the humidifier.

[0058] Test Example 2 The B16 melanoma cell line was purchased from the National Institute of Biomedical Innovation (JCRB0202, Osaka, Japan) and cultured in Dulbecco's Eagle's minimum essential medium supplemented with 10% fetal bovine serum. After the cells seeded in a 96-well plate became confluent, they were treated with the humidifier. After 72 hours, the cells were solubilized with DMSO, and the absorbance at 490 nm was measured with a microplate reader.

[0059] From the results of this experiment, it was confirmed that the humidifier had no effect on melanin synthesis in melanoma.

[0060] Test Example 3 Mouse melanoma B16 cells were adjusted to 1×10 810 ml of cell lysate (0.1 M Tris, 1% NP-40, 0.01% SDS, pH 7.5) was added to the cells, mixed, and disrupted by pipetting. It was left on ice for 30 minutes. The cell lysate was centrifuged (1,000 g, 10 minutes), and the supernatant was collected and centrifuged again (20,000 g, 10 minutes). The pellet was washed with PBS(-) and centrifuged (20,000 g, 10 minutes), and then suspended in 1 ml of autophagy induction medium to prepare a melanosome extraction pellet. 6 μl (equivalent to 7.5×10 5 cells) of melanosomes were added to each well.

[0061] HaCaT cells were seeded in a 96-well plate. When the cells became confluent, melanosomes prepared from the B16 melanoma cell line were added daily along with medium replacement. After 4 days, treatment was performed with a humidifier using autophagy induction medium. After 72 hours, the cells were solubilized with DMSO, and the absorbance at 490 nm was measured using a microplate reader.

[0062] From the results of this experiment, it was confirmed that the addition of a humidifier improved the digestive activity of melanosomes in keratinocytes in a concentration-dependent manner.

[0063] Test Example 4 The effect of 3-methyladenine (3MA), an autophagy inhibitor, on the melanin digestive activity of keratinocytes was examined. It was performed in the same manner as Test Example 3 except that 3MA was added.

[0064] The results are shown in Figure 1. 5 mM 3MA reversed the melanin removal effect of the humidifier (Hu) on HaCaT cells that had taken up melanosomes. This indicates that melanin digestion in keratinocytes occurred by autophagy.

[0065] Test Example 5 The effect of metformin (MET), a decoupling agent, on the melanin digestive activity of keratinocytes was examined. It was performed in the same manner as Test Example 3 except that MET was added.

[0066] The results are shown in Figure 2. Neither 500 μM MET nor 0.0001% Hu showed significant melanosome digestion activity alone, but when both were treated simultaneously, a significant effect was shown.

[0067] Test Example 6 The effects of fulvic acid and minerals on the melanin digestion activity of keratinocytes were investigated. The experiment was conducted in the same manner as in Test Example 3 except that fulvic acid and minerals were added. The minerals were obtained by subjecting humicules to strong heat treatment in an electric furnace (600 °C, 12 hours × 2 times) and repeating the process until the product reached 200 g.

[0068] The results are shown in Figure 3. No melanosome digestion activity was observed even when 0.0001% of the fulvic acid (FA) standard product purchased from the Japanese Humic Substances Society or 0.01% of the minerals obtained by burning organic substances from humicules was exposed, but when both were treated simultaneously, a significant effect was shown.

Claims

**Claim 1** An autophagy inducer comprising a composition containing fulvic acid. **Claim 2** The autophagy inducer according to claim 1, which induces autophagy in keratinocytes. **Claim 3** The autophagy inducer according to claim 1 or 2, which is used for whitening and / or suppressing epidermal hyperplasia. **Claim 4** The autophagy inducer according to any one of claims 1 to 3, wherein the composition containing fulvic acid has a decoupling effect.

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